This invention relates to a novel anti-antibody, or secondary antibody, reagent for use in an immunoassay procedure, to a method for its production and to its use in immunoassays, including both simplex assays (assays for a single analyte) and multiplex assays (assays carried out simultaneously for a plurality of analytes), particularly to automated assays of that type
Very generally, the immunoassays are performed to determine whether an analyte, or members of a plurality of analytes, are present in a sample, typically a liquid sample, and also may be used to determine, at least to some degree, the amount of such analytes (if any) in the sample.
Immunoassays of this type are used for numerous purposes, for instance as to determine whether any drugs, proteins, antigens, allergens, pollutants, or other substances are present in a liquid sample, and/or are present in an amount greater or lower than some quantity. The sample could be, for instance, a sample of a human or other mammalian body fluid such as blood, serum, urine, sweat, saliva or stool samples, a liquid sample obtained in a research effort, laboratory procedure or the like, or a sample of water or other liquid material obtained from natural or industrial sources, and the like.
In competitive immunoassays, generally, the sample is placed in contact, or is incubated with, a first antibody (in a simplex assay), or a plurality of first or primary antibodies (in a multiplex assay) for the analytes whose presence and/or amount in the sample is sought to be ascertained, together with molecules of the analyte or analytes to be determined bound or coupled to a solid substrate. An assay is then carried out. The assay typically will be a heterogeneous assay, but could be a homogeneous assay. The solid substrate may have a planar or similar surface, such as a flat plate, a microfluidic device or the walls of wells of a multi-well plate, or it may comprise microparticles or beads. Coupled material is then contacted or incubated with an anti-antibody (in a simplex assay) or a plurality of anti-antibodies (in a multiplex assay) for the primary antibody. The anti-antibodies are tagged or labeled with a detectable label such as a fluorophore, chromophore, radioactive label, etc. that can be detected either visually or, preferably, with an instrument. An appropriate instrument such as a plate reader in the case of a multi-well plate enzyme immunoassay or a flow cytometer when microparticles or beads are used, is employed to detect the label and identify which (if any) analytes are present.
Other immunoassays are used to detect antibodies in serum or plasma that are the result of autoimmune conditions, allergic reactions, or exposure to an infectious agent. In such immunoassays, antigens or a plurality of antigens corresponding to the antibodies of interest are immobilized on a solid surface. The solid substrate may have a planar or similar surface, such as a flat plate, a microfluidic device or the walls of wells of a multi-well plate, or it may comprise microparticles or beads. The specimen is contacted with the surface, and, during an ensuing incubation, antibodies in the specimen, if present, are allowed to bind the immobilized antigens. Coupled material is then contacted or incubated with an anti-antibody (in a simplex assay) or a plurality of anti-antibodies (in a multiplex assay) for the antibody in the specimen. The anti-antibodies are tagged or labeled with a detectable label such as a fluorophore, chromophore, radioactive label, etc. that can be detected either visually or, preferably, with an instrument. An appropriate instrument such as a plate reader in the case of a multi-well plate enzyme immunoassays, or a flow cytometer when microparticles or beads are used, is employed to detect the label and identify which (if any) analytes are present.
Another type of assay can be used to detect a variety of analytes provided that they can be bound simultaneously by two antibodies. This “sandwich” technique consists of incubating the specimen with a first antibody coupled to a solid phase, and a second antibody. If the analyte is present, it is bound by both antibodies, the complexes are separated from the reaction mixture, and the bound second antibody is detected. Typically, the second antibody is labeled with an enzyme, fluorophore, or other species that can be detected directly. Alternatively, a third antibody or other ligand could be used. For example, the second antibodies, or plurality of second antibodies in a multiple assay, could be biotinylated, then detected using streptavidin labeled with an appropriate fluorophore. As another example, the second antibodies could be produced in a different species than was used for the first antibodies (e.g. mouse vs. rabbit) and an appropriately labeled antibody to mouse IgG would detect all bound second antibodies in the mixture.
Assays such as those described above benefit from using labeled anti-antibody reagents that are easily produced, whose optimal working concentration can be easily determined and adjusted, and the use of which results in a robust assay. A robust assay uses reagents in excess or near excess when possible in order to avoid analytical error due to variability in volume of reagent dispensed, time of incubation, dilution by residual wash fluid, and other factors that are rendered less intrusive if concentration of the reagent is not critical. Labeled anti-antibody reagents can be used in excess, but achieving an excess level of anti-antibody activity can lead to generation of excessive amounts of signal.